Cholesterol and Morpholine Grafted Cationic Amphiphilic Copolymers for miRNA-34a Delivery

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00228

miR-34a is a master tumor suppressor playing a key role in the several signaling mechanisms involved in cancer. However, its delivery to the cancer cells is the bottleneck in its clinical translation. Herein we report cationic amphiphilic copolymers graft

Full text of DOI:10.1021/acs.molpharmaceut.8b00228

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Article
Cholesterol and Morpholine Grafted Cationic
Amphiphilic Copolymers for miRNA-34a Delivery
Saurabh Sharma, Samrat Mazumdar, Kishan S Italiya, Tushar
Date, Ram I. Mahato, ANUPAMA MITTAL, and Deepak Chitkara
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00228 • Publication Date (Web): 10 May 2018
Downloaded from http://pubs.acs.org on May 15, 2018
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Cholesterol and Morpholine Grafted Cationic Amphiphilic Copolymers for miRNA-
34a Delivery
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Saurabh Sharma1, Samrat Mazumdar1, Kishan S. Italiya1, Tushar Date1, Ram I. Mahato2,
Anupama Mittal1, Deepak Chitkara1*
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1Department of Pharmacy,
Birla Institute of Technology and ScienceꢀPilani (BITS),
Pilani Campus, Vidya Vihar, Pilani ꢀ 333031 (Rajasthan) India.
2Department of Pharmaceutical Sciences,
College of Pharmacy,
University of Nebraska Medical Center
986125 Nebraska Medical Center
Omaha, NE 68198ꢀ6125
*Corresponding author
Deepak Chitkara, Ph.D.
Department of Pharmacy,
Birla Institute of Technology and ScienceꢀPilani (BITS),
Pilani Campus, VidyaVihar, Pilani ꢀ 333 031 (Rajasthan) India.
Eꢀmail: deepak.chitkara@pilani.bitsꢀpilani.ac.in
Contact no: 01596 515 835 (O), +91 9660 456 009 (M)
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ABSTRACT
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miRꢀ34a is a master tumor suppressor playing a key role in the several signalling
mechanisms involved in cancer. However its delivery to the cancer cells is the bottleneck
in its clinical translation. Herein we report cationic amphiphilic copolymers grafted with
cholesterol (chol), N,Nꢀdimethyldipropylenetriamine (cation chain) and 4ꢀ(2ꢀ
aminoethyl)morpholine (morph) for miRꢀ34a delivery. The copolymer interacts with
miRꢀ34a at low N/P ratios (~2/1) to form nanoplexes of size ~108 nm and a zeta potential
~ +39 mV. In vitro studies in 4T1 and MCFꢀ7 cells indicated efficient transfection
efficiency. The intracellular colocalization suggested that the copolymer effectively
transported the FAM labeled siRNA into the cytoplasm within 2 h and escaped from the
endo/lysosomal environment. The developed miRꢀ34a nanoplexes inhibited the breast
cancer cell growth as confirmed by MTT assay wherein 28% and 34% cancer cell
viability was observed in 4T1 and MCFꢀ7 cells, respectively. Further, miRꢀ34a
nanoplexes possess immense potential to induce apoptosis in both cell lines.
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Key words: Nanoplexes; miRꢀ34a; Amphiphilic cationic polymer; Anticancer therapy
INTRODUCTION
miRNAs are endogenous, short, nonꢀcoding, 20ꢀ25 base pair long
oligonucleotides involved in gene expression regulation via mRNA degradation or
translation repression.1 Their abberant expression has been implicated in several diseases
including cancer wherein either oncogenic miRNAs are overexpressed or tumor
suppressor miRNAs are underexpressed.2 Among various miRNAs, miRNAꢀ34a (miRꢀ
34a) is known as master tumor suppressor which is transcriptionally activated by p53 and
plays a key role in cell proliferation, apoptosis and metastasis.2 Its role has been
implicated in several cancers including breast cancer,3 prostate cancer,4 lung cancer5 and
pancreatic cancer6 wherein, it suppresses cell proliferation and induces apoptosis by
targeting several mRNAs including LMTK3,7 SIRT1,8 cSRC9 and CD44.10 Although the
potential of miRNA therapeutics is now well established, however to achieve its optimal
benefit in clinic, several biopharmaceutical issues need to be addressed. Physicochemical
properties of miRNAs such as hydrophilicity, high molecular weight and negative charge
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makes them impermeable to the cellular membranes.11 Further naked miRNAs are easily
filtered through the glomerulus and are unstable in biological milieu due to presence of
RNAses thus making their systemic administration impractical. Several nonꢀviral vectors
including lipoplexes,12 cationic solid lipid nanoparticles,13 polyplexes,14 micelleplexes15
and dendriplexes16 have been reported for miRNA delivery. Use of cationic amphiphilic
copolymers forms an attractive strategy for complexation with negatively charged
miRNA. In a study by Zhang et al. poly(ethylene glycolꢀbꢀlactideꢀbꢀarginine) copolymer
was used for preparing reducible nanoformulation for antiꢀmiRꢀ21 delivery to
cerebrospinal fluid.15 In another study, dual stimuliꢀsensitive mixed polymeric micelles
efficiently delivered miRꢀ34a and doxorubicin in tumor tissue.17 One major advantage of
these systems is that their block copolymer structure and composition could be suitably
modified to overcome the hurdles of miRNA delivery. Previously, we have also shown
that cationic amphiphilic mPEGꢀpolycarbonate based copolymers could deliver miRꢀ205
and gemcitabine to pancreatic cancer cells in vitro and in vivo.18 In another study, a
hedgehog inhibitor, GDCꢀ0449, and miRꢀlet7b was coꢀdelivered using these micelles for
treating pancreatic cancer.19
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To further improve the therapeutic outcome of miRNA delivery, important
aspects including efficient transfection into the cells followed by their endosomal escape
and release of the miRNA in the cytoplasm needs to be controlled. Attempts have been
made to improve the delivery by incorporating cholesterol in the carrier system.
Cholesterol conjugated chitosan has been used for delivering small molecules including
curcumin, paclitaxel and doxorubicin.20ꢀ23 Huang et al. prepared poly(βꢀamino esters) and
PEG based triblock copolymers grafted with cholesterol for doxorubicin delivery.24 In a
recent study, incorporation of cholesterolꢀpoly(2ꢀ(dimethylamino)ethyl methacrylate) in
lecithin liposomes showed enhanced internalization with no coꢀlocalization with acidic
compartments in HEKꢀ293 cells.25 Incorporation of a weak base like imidazole group or
histamine and guanidine in the polymer have been shown to impart endosomal escape by
proton sponge effect. Frere et al. showed that morpholine functionality in a polycarbonate
based polymer confers buffering ability and proton sponge effect to the polymer.26
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Herein we report amphiphilic copolymers containing cholesterol, morpholine, and
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cationic pendant groups (N,Nꢀdimethyldipropylenetriamine) on the polycarbonate
backbone that could form stable nanoplexes with miRꢀ34a at low N/P ratios. Nanoplexes
were prepared by film hydration method or double emulsion method followed by their
thorough characterization for size, zeta potential and complexation ability. Flow
cytometric analysis was performed to study the transfection efficiency of FAMꢀsiRNA
loaded nanoplexes in 4T1 (murine) and MCFꢀ7 (human) breast cancer cells. Further coꢀ
localization of the nanoplexes with the endo/lysosomes was determined by confocal
microscopy. Hemocompatibility of the nanoplexes was determined by hemolysis assay.
Cytotoxicity and apoptosis assessment of miRꢀ34a containing nanoplexes were then
performed in 4T1 and MCFꢀ7 breast cancer cells by MTT assay and annexinꢀ
V/propidium staining, respectively.
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EXPERIMENTAL SECTION
Materials
miRNAꢀ34aꢀ5p mimic, mirVana™ miRNA Mimic #1 Negative control and
LysoTracker Red DNDꢀ99 were purchased from ThermoFischer scientific
(Massachusetts, USA). Lipofectamineꢀ2000®, Fetal Bovine Serum (FBS), Dulbecco’s
Modified Eagle Medium (DMEM), Annexin V Alexa fluor® 488 conjugate and TrypLE
were obtained from ThermoFisher Scientific (USA). FAMꢀsiRNA was purchased from
the GeneCust Europe (Luxembourg). Bis(hydroxymethyl) propionic acid, benzyl
bromide, tin(II) 2ꢀethylhexanoate, methoxy poly(ethylene glycol) (mPEG, 5000 Da), 4ꢀ
(2ꢀaminoethyl)morpholine, cholesterol, N,Nꢀdimethyldipropylenetriamine (DP) and
propidium iodide (PI) were purchased from Sigma Aldrich (St. Louis, MO). DLꢀLactide
and N, Nꢀdiisopropylethylamine (DIPEA) were purchased from TCI Chemicals (Japan).
All other chemicals and reagents were of analytical grade and purchased from local
vendors.
Synthesis of cationic amphiphilic copolymers
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A series of cationic amphiphilic copolymers with different ligands (Table 1) on
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the hydrophobic segment were synthesized using a multiꢀstep reaction consisting of
synthesis of cyclic monomer, 2ꢀmethylꢀ2ꢀbenzyloxycarbonylpropylene carbonate (MBC),
synthesis of mPEGꢀbꢀP(CBꢀcoꢀLA) diblock copolymer followed by its reduction and
coupling of ligands including N,Nꢀdimethyldipropylenetriamine, cholesterol and/or 4ꢀ(2ꢀ
aminoethyl)morpholine by EDC/HOBT coupling chemistry as shown in Figure 1.
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Figure 1. Synthesis of amphiphilic copolymers grafted with N,Nꢀdimethyldipropylenetriamine
(cation chain), cholesterol and 4ꢀ(2ꢀaminoethyl) morpholine.
Briefly, cyclic monomer, MBC was synthesized in a twoꢀstep reaction, wherein a mixture
of 2,2ꢀbis(hydroxymethyl) propionic acid and potassium hydroxide was reacted with
benzyl bromide in dimethyl formamide (DMF) at 100°C for 15 h. Work up of the crude
product was performed followed by recrystallization from toluene to yield a pure
intermediate product, benzylꢀ2,2ꢀbis(methylol)propionate. In the second step, benzyl 2,2ꢀ
bis(methylol)propionate (in dichloromethaneꢀpyridine mixture) was reacted with
triphosgene to obtain MBC as a crude product, which was further recrystallized from
ethyl acetate to obtain MBC as white crystals.27 Amphiphilic copolymer viz. mPEGꢀbꢀ
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P(CBꢀcoꢀLA), was synthesized by ring opening polymerization of mPEG with MBC and
DLꢀlactide in presence of tin(II) 2ꢀethylhexanoate (10 mol% of mPEG) as catalyst. The
reaction was allowed to continue for 20 h at 130°C under nitrogen. Crude polymer
obtained was dissolved in chloroform and purified by precipitation using diethylether and
isopropyl alcohol followed by drying under vacuum. The protective benzyl groups from
mPEGꢀbꢀP(CBꢀcoꢀLA) copolymer were removed by catalytic hydrogenation using Pd/C
under 45 psi pressure to obtain mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀLA) with free carboxyl
pendant groups on the polymer backbone.28
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Carbodiimide/Nꢀhydroxybenzotriazole (HOBt) coupling was used to graft 4ꢀ(2ꢀ
aminoethyl)morpholine, cholesterol and/or N,Nꢀdimethyldipropylenetriamine on the free
carboxyl pendant groups of mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀLA) copolymer to yield four
cationic copolymers (i.e. containing only cation chain, cation chain + morpholine group,
cation chain + cholesterol, and cation chain + cholesterol + morpholine group) and named
as P1, P2, P3 and P4, respectively (Table 1). Crude polymers were subsequently purified
by precipitation in isopropanol and diethyl ether followed by drying under vacuum. These
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copolymers were then characterized by H NMR and 13C NMR to determine the
structure, composition and molecular weight.28 For this purpose, copolymers were
dissolved in deuterated chloroform (CDCl3) except for mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀ
LA) which was dissolved in DMSOꢀd6 and analyzed using Bruker (400 MHz) NMR
spectrometer. Elemental composition of synthesized copolymers was determined using
Elementar Vario EL III CꢀHꢀN analyzer (Sophisticated Test and Instrumentation Centre
(STIC), Cochin University of Science and Technology, Kerala, India) which is based on
combustion analysis. An accurately weighed sample was burned in an excess of oxygen
followed by collection of the combustion products such as carbondioxide, water and
nitric oxide by different traps. Masses of these combustion products were used to
calculate the percentage of C, H and N present in the copolymers.
Formulation of miR-34a nanoplexes
The nanoplexes of all four copolymers with miRꢀ34a were prepared by two
methods i.e. film hydration (FH) method and double emulsion solvent evaporation
method (DE).30,31 In film hydration method, copolymer (10 mg) was dissolved in
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dichloromethane (1 mL) followed by evaporation of the solvent under reduced pressure
to obtain a thin film. The film was further redispersed with DNAse/RNAse free purified
water containing miRꢀ34a (200 pmole; 1 mL). The formed nanoplexes were allowed to
stand for 30 min at room temperature followed by centrifugation at 2000 rpm for 5 min at
4°C. In DE method, the internal aqueous phase consisting of miRꢀ34a solution (40 µL; 5
pmole/µL) in DNAse/RNAse free purified water was first prepared. The organic phase
was prepared by dissolving copolymer (10 mg) in dichloromethane (0.5 mL). The
internal aqueous phase was added drop wise into organic phase and sonicated for 30
seconds on bath sonicator (Ultrasonic, Macroscientific) to form primary water in oil (w/o)
emulsion which was subsequently added to 1 mL of DNAse/RNAse free purified water
and kept for bath sonication for 1 min to obtain w/o/w double emulsion. The resulting
double emulsion was stirred at room temperature for 2 h (to evaporate the organic
solvent) centrifuged at 2000 rpm for 5 min at 4°C and filtered using 0.22 µm membrane
filter. For particle size, polydispersity index and zeta potential analysis, nanoplexes were
taken at a concentration equivalent to 1 mg/mL of polymer in DNase/RNase free purified
water and analyzed using Zetasizer (Malvern Nano ZS ).
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Agarose gel retardation assay
Agarose gel retardation assay was performed to determine the N/P ratio for
effective complexation between synthesized cationic copolymers and miRꢀ34a as
reported earlier.18 Briefly, nanoplexes were prepared by double emulsion method at N/P
ratio ranging from 1/1 to 16/1 and loaded on a agarose gel (2% w/v) containing 0.5
µg/mL ethidium bromide (EtBr) and run for 30 min at 90 V in 0.5X TrisꢀBorateꢀEDTA
(TBE) buffer. The electrophoretic mobility of miRꢀ34a was visualized on a Gel Doc™
XR+ Gel Documentation system.
Hemocompatibility assay
Pooled blood sample (2 mL) was collected from swiss albino mice and mixed
with EDTA to prevent coagulation. RBCs were obtained from the blood by centrifugation
at 1500 rpm at 4ºC for 15 min followed by washing with normal saline solution.31 These
washed RBCs were resuspended in 5 mL of normal saline to obtain RBCs dispersion.
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P4/miRꢀ34 nanoplexes, blank nanoplexes and distilled water (positive control) were
added to 1 mL of RBCs dispersion and incubated at 37°C for 1 h followed by
centrifugation at 2000 rpm for 5 min. The supernatant was collected and analyzed using
UV spectrophotometer at 415 nm and the percentage hemolysis in the samples was
determined considering the percent hemolysis induced by distilled water as 100%.
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Cell culture studies
4T1 cells were obtained as a kind gift from Dr. Avinash Bajaj (Associate
Professor, Regional Centre for Biotechnology, Haryana (NCR Delhi), India) and MCFꢀ7
cells were obtained from National Centre for Cell Science, Pune. Both cell lines were
cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS; HyClone, Logan, UT) and 1% antibiotics and kept in an incubator at
37°C/ 5% CO2.
Transfection efficiency
FAMꢀsiRNA nanoplexes were used for studying the transfection efficiency by
flow cytometry as reported earlier.32 Briefly, 4T1 and MCFꢀ7 cells were seeded at a cell
density of 1×105 cells/well in 6 well cell culture plate and allowed to adhere for 24 h.
Prior to treatment with the nanoplexes, culture media was replaced with OptiꢀMEM
medium for 1 h. Cells were treated then with FAMꢀsiRNA nanoplexes for 4 h. Naked
FAMꢀsiRNA and lipofectamineꢀ2000®/FAMꢀsiRNA lipoplexes were kept as negative
and positive control, respectively. After treatment, cells were washed thrice with PBS,
trypsinized, centrifuged at 2000 rpm/5 min at 4ºC and flow cytometric analysis was
performed on Cytoflex (Beckman Coulter, USA). The FAMꢀsiRNA was excited with an
argon laser (488 nm), and fluorescence was measured at 525 nm. Data was analyzed
using CytExpert software.
Endo/lysosomal escape
4T1 and MCFꢀ7 cells (1×105 cells/well) were seeded onto coverslips in a 6 well
cell culture plates. After 24 h, cells were washed with PBS (twice) and media was
replaced with OptiꢀMEM followed by treatment with FAMꢀsiRNA nanoplexes or
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lipofectamineꢀ2000®/FAMꢀsiRNA lipoplexes. Cells were then incubated with
LysoTracker Red DND 99 (1µM) for 20 min for endo/lysosome labelling. Cells were
washed with PBS, fixed in paraformaldehyde (4%) for 10 min and counterstained with
DAPI (for nucleus staining; 5µg/mL)). The cells were observed at 100 X (Plan SꢀApo,
NA 1.4, WD 0.12 mm) (DIC) under confocal laser scanning microscope (Olympus IX71,
DP71, Tokyo, Japan).16 The colocalization of FAMꢀsiRNA with endo/lysosomes was
analysed using BioImage XD software wherein scattergram was plotted between the
FAMꢀsiRNA fluorescence (green) and lysosomal fluorescence (red) and Costes method
was used for calulating thresholds and statistics.34, 35
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Cytotoxicity studies
Cytotoxicity of miRꢀ34a nanoplexes was assessed as reported earlier.37,38 Briefly,
cells (4T1 or MCFꢀ7) were seeded in 96 well plates (5x103 cells/well) and allowed to
adhere for 24 h followed by treatment with nanoplexes containing 20 picomole of miRꢀ
34a. Negative control (NC) nanoplexes (containing mirVana™ miRNA Mimic #1
Negative control, a random sequence miRNA mimic molecule) and blank nanoplexes
were kept as controls. After 48 h, cell viability was analysed using 3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay. Absorbance was
recorded by microplate reader (BioTek Epoch) at 560 nm and corrected for the cell debris
by subtracting absorbance at 630 nm. The percentage cell viability was determined using
the following formula:
% Cell viability
=ꢀ(ꢁꢂꢃꢄꢅꢂꢆꢇꢈꢉ ꢄꢊ ꢃꢆꢋꢌꢍꢉ ꢎꢉꢍꢍꢃ/ꢁꢂꢃꢄꢅꢂꢆꢇꢈꢉ ꢄꢊ ꢈꢄꢇꢏꢅꢄꢍ ꢎꢉꢍꢍꢃ)
× 100
Apoptosis assay
Annexin V, Alexa Fluor™ 488 conjugate was used to detect and quantify
apoptosis induced by miRꢀ34a nanoplexes using flow cytometry as per manufacturer’s
protocol. Briefly, 4T1 or MCFꢀ7 cells were seeded (1x106 cells/well) in a 6 well cell
culture plate. After 24 h, the media was replaced with fresh media containing blank
nanoplexes, miRꢀ34a nanoplexes or NC miRNA nanoplexes and incubated with cells for
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24 h at 37°C/5% CO2. After treatment, cells were washed with PBS, trypsinized,
centrifuged at 2000 rpm/5 min at 4°C, resuspended in 1X binding buffer and stained with
Annexin V conjugate (5 ꢁl) and propidium iodide (PI) (10 ꢁl). Flow cytometric analysis
was performed on Cytoflex (Beckman Coulter, USA) and data were interpreted using
CytExpert software.13, 39
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Statistical analysis
Data are presented as the mean ± standard deviation. The difference between any
two or more groups was determined by analysis of variance. P< 0.05 was considered to
be statistically significant.
RESULTS
Synthesis and characterization of monomers and copolymers
For delivery of miRꢀ34a, we have synthesized amphiphilic cationic copolymers
grafted with ligands including 4ꢀ(2ꢀaminoethyl)morpholine, cholesterol and N,Nꢀ
dimethyldipropylenetriamine onto the hydrophobic block (Table 1). A cyclic carbonate
monomer, MBC (2ꢀmethylꢀ2ꢀbenzyoxycarbonyl propylene carbonate) was synthesized as
reported earlier.27 1H NMR of MBC showed peaks corresponding to δ 1.2 (CH3, s, 1H) , δ
4.3 (CH2, d, 2H) and 4.7 (CH2, d, 2H) , δ 5.2 (CH2, s, 2H) and δ 7.3 (C6H5, m, 5H). Mass
spectroscopy results showed a molecular ion (sodium adduct) peak at 273 m/z and its
(benzylic) daughter ion peak at 90 m/z indicating the successful synthesis of the
monomer. Further ring opening polymerization of mPEG with DLꢀlactide and MBC in
the presence of tin(II) 2ꢀethylhexanoate yielded mPEGꢀbꢀP(CBꢀcoꢀLA) copolymer with
1
80% practical yield. Figure 2 A shows H NMR (400 MHz,CDCl3) of synthesized
copolymer mPEGꢀbꢀP(CBꢀcoꢀLA) that showed peaks for mPEG protons at δ 3.56 (CH2,
m, 4H), for 2ꢀmethylꢀ2ꢀbenzyoxycarbonylꢀpropylene carbonate (CB) unit protons at δ
1.24 (CH3, s, 3H), δ 4.3 (CH2, m, 4H ), δ 5.2 (CH2, s, 2H), δ 7.3 (C6H5, m, 5H) and for
lactic acid (LA) protons at δ 1.6 (CH3, s, 3H) and δ 5.2 (CH, q, 1H). Further the absence
of two peaks (of MBC monomer) at δ 4.3 and δ 4.7 and presence of one proton peak at δ
4.3 (CH2, m, 4H) indicated the successful ring opening polymerization. Further 13C
NMR (100 MHz, CDCl3) (figure 2 B) of mPEGꢀbꢀP(CBꢀcoꢀLA) showed peaks at δ
171.9, 169.6, 169.4, 169.3, 154.3, 154, 135.3, 128.6, 128.6, 128.4, 128.3, 128.1, 127.9,
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71.5, 70.6, 69.2, 69, 68.6, 67.1, 66.1, 46.6, 17.4 and 16.7. mPEGꢀbꢀP(CBꢀcoꢀLA)
copolymer was catalytically hydrogenated to obtain mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀLA)
with free carboxyl pendant groups on the polymer backbone with > 90% practical yield.
1H NMR (400 MHz, DMSOꢀd6) (figure 2 C) of mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀLA)
showed peaks at δ 12ꢀ13 (COOH, s, 1H) and absence of phenylic protons. The broad
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peak corresponding to carboxylic acid protons was observed in the H NMR taken in
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DMSOꢀd6 and was thus used as a solvent of choice for this polymer. Its C NMR (100
MHz, CDCl3) (figure 2 D) showed peaks at δ 169.6, 169.4, 128.6, 128.3, 128.2, 127.9,
70.6, 69.2, 69.0, 67.9, 46.6, 25.6 and 16.7.
Table 1. Characterization of cationic amphiphilic copolymers.
Polymer
code
Cationic Copolymers
Elemental Analysis
1H NMR
Sample
Weight
(mg)
Molecular
weight (Da)
C%
H%
N%
P1
P2
P3
P4
mPEGꢀbꢀP(CB82ꢀ{gꢀCation
chain11}ꢀcoꢀLA115)
30,209
24,342
43,523
35,436
11.98
4.95
4.28
50.39
45.63
46.30
9.78
7.79
7.28
3.76
7.68
6.48
mPEGꢀbꢀP(CB44ꢀ{gꢀCation
chain13; gꢀMorph5}ꢀcoꢀLA110)
mPEGꢀbꢀP(CB78ꢀ{gꢀCation
chain36; gꢀChol30}ꢀcoꢀLA110)
mPEGꢀbꢀP(CB65ꢀ{gꢀCation
chain11; gꢀChol19; gꢀMorph6;}ꢀ
coꢀLA120)
2.56
45.73
5.36
7.55
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Figure 2. (A and B) H NMR and C NMR of the mPEGꢀbꢀP(CBꢀcoꢀLA), (C and D) H NMR and
13C NMR of the mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀLA).
Figure 3. 1H NMR of the synthesized cationic copolymers. P1: mPEGꢀbꢀP(CBꢀ{gꢀCation chain}ꢀ
coꢀLA), P2: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀMorph}ꢀcoꢀLA), P3: mPEGꢀbꢀP(CBꢀ{gꢀCation
chain; gꢀChol}ꢀcoꢀLA) and P4: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀChol; gꢀMorph}ꢀcoꢀLA).
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Figure 4. 13C NMR of the synthesized cationic copolymers. P1: mPEGꢀbꢀP(CBꢀ{gꢀ
Cation chain}ꢀcoꢀLA), P2: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀMorph}ꢀcoꢀLA), P3:
mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀChol}ꢀcoꢀLA) and P4: mPEGꢀbꢀP(CBꢀ{gꢀCation
chain; gꢀChol; gꢀMorph}ꢀcoꢀLA).
Carbodiimide/Nꢀhydroxybenzotriazole (HOBt) coupling was used to graft 4ꢀ(2ꢀ
aminoethyl) morpholine, cholesterol and N,Nꢀdimethyldipropylenetriamine on to the free
carboxyl pendant groups of mPEGꢀbꢀP(CBꢀ{gꢀCOOH}ꢀcoꢀLA) copolymer to yield a
1
series of cationic copolymers as shown in Table 1. H NMR analysis showed peaks
corresponding to protons of mPEG, LA and CB units as given above in addition to the
peaks of functional moieties such as cholesterol (at δ 0.8ꢀ1.0 (CH3, s, 6H), δ 4.5 (CH, q,
1H), δ 5.4 (CH, d, 1H)), N,Nꢀdimethyldipropylenetriamine (at δ 2.1 (NH, s, 1H), δ 7.4
(Amidic NH, s, 1H)) and 4ꢀ(2ꢀaminoethyl) morpholine (at δ 3.7 (CH2, m, 4H)) thus
indicating the effective coupling of ligands. 13C NMR (100 MHz, CDCl3) of the
synthesized copolymers is shown in figure 4. Copolymer P1 showed peaks at δ (ppm)
171.8, 169.5, 169.3, 135.4, 128.6, 128.4, 128.1, 127.9, 70.6, 69.2, 69.0, 67.1, 67.0, 65.9,
46.6, 25.3, 17.4, 16.7 and 15.3. Copolymer P2 showed peaks at δ (ppm) 176.6, 171.9,
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135.3, 128.6, 128.4, 128.1, 127.9, 71.9, 71.2, 68.9, 67.0, 64.3, 58.9, 46.6 and 16.7.
Copolymer P3 showed peaks at δ (ppm) 169.6, 169.4, 169.3, 169.1, 135.3, 128.6, 128.3,
128.0, 127.9, 70.6, 69.2, 69, 65.8, 46.6, 25.1, 16.7 and 15.3, and copolymer P4 showed
peaks at δ (ppm) 171.9, 169.7, 169.6, 169.3, 128.6, 128.4, 128.1, 128.0, 71.6, 70.6, 69.0,
67.0, 65.8, 46.6, 17.5, 16.7 and 15.3. The molecular weight of the copolymers (P1, P2, P3
and P4) were determined using 1H NMR and found to be 30209, 24342, 43523 and 35436
Da respectively. Total carbon, hydrogen and nitrogen content of the cationic copolymers
(P1, P2, P3 and P4) was determined by elemental analysis as shown in Table 1.
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Table 2. Characterization of miRꢀ34a nanoplexes prepared using cationic amphiphilic copolymers.
Size
(d.nm)
Zeta potential
(mV)
S. No
Polymer
P1
PDI
180 ± 6.38
120 ± 7.42
112 ± 7.89
108 ± 4.56
0.187
12 ± 0.74
31 ± 1.07
27 ± 0.94
39 ± 0.84
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P3
P4
0.170
0.263
0.112
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3
4
Characterization of miRNA-34a nanoplexes
miRNA-34a nanoplexes prepared by DE method exhibited a mean particle size of
<200 nm with positive zeta potential ranging from 12 to 39 mV and unimodal distribution
showing bell shape curve with polydispersity index (PDI) ranging from 0.112-0.263
(Figure 5 and Table 2). In case of film hydration method, nanoplexes exhibited a
particle size <200 nm but the polydispersity index (PDI) was high (0.58-0.632) with
bimodal particle size distribution.
Agarose gel retardation assay
The complexation ability of copolymers with miRꢀ34a was performed by agarose
gel retardation assay. The assay was based on the restricted mobility of miRꢀ34a due to
binding with cationic polymers thus resulting in disappearance of miRNA bands in the
agarose gel. miRꢀ34a nanoplexes were prepared by DE method at N/P ratio ranging from
1/1 to 16/1. The results demonstrated that the electrophoretic mobility of miRꢀ34a was
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fully retarded at N/P of 2/1 for miRꢀ34a nanoplexes prepared using P4 copolymer
indicating complete complex formation with miRꢀ34a while miRꢀ34a nanoplexes of P1,
P2 and P3 showed complex formation at N/P of 8/1, 16/1 and 8/1, respectively (Figure
5C).
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Hemocompatibility assay
Figure 5. (A) Particle size and (B) Zeta potential of the nanoplexes prepared using cationic
polymers, (C) miRNA binding ability of cationic polymers investigated by agarose gel
electrophoresis. (D) Hemolysis assay of nanoplexes prepared using P4 copolymer. P1:
mPEGꢀbꢀP(CBꢀ{gꢀCation chain}ꢀcoꢀLA), P2: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀMorph}ꢀ
coꢀLA), P3: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀChol}ꢀcoꢀLA) and P4: mPEGꢀbꢀP(CBꢀ{gꢀ
Cation chain; gꢀChol; gꢀMorph}ꢀcoꢀLA).
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The hemocompatibility assay was performed to study whether the designed
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formulation carries any RBCs membrane destabilization potential that could be correlated
to the hemotoxicity. In hemolysis test the nanoplexes were incubated with the RBCs for 1
h followed by determination of hemoglobin content by UV spectroscopy. It was observed
that the blank nanoplexes and miRꢀ34a nanoplexes showed 3.26% and 2.86% hemolysis
respectively, as compared to double distilled water that showed 100% hemolysis (Figure
5D).
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In vitro transfection efficiency
To demonstrate the ability of the nanoplexes to transfect 4T1 and MCFꢀ7 breast
cancer cells, we performed the flow cytometry analysis after incubation of cancer cells
with FAMꢀsiRNA/nanoplexes. Lipofectamineꢀ2000®/FAMꢀsiRNA lipoplexes was taken
Figure 6. Transfection efficiency of the FAMꢀsiRNA in 4T1 cells by flow cytometry. A) Naked
FAMꢀsiRNA, B) Lipofectamineꢀ2000®/FAMꢀsiRNA, C) P1/FAMꢀsiRNA, D) P2/FAMꢀsiRNA, E)
P3/FAMꢀsiRNA and F) P4/FAMꢀsiRNA. P1: mPEGꢀbꢀP(CBꢀ{gꢀCation chain}ꢀcoꢀLA), P2:
mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀMorph}ꢀcoꢀLA), P3: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀChol}ꢀ
coꢀLA) and P4: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀChol; gꢀMorph}ꢀcoꢀLA).
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Figure 7. Transfection efficiency of FAMꢀsiRNA in MCFꢀ7 cells by flow cytometry. A) Naked FAMꢀ
siRNA, B) Lipofectamineꢀ2000®/FAMꢀsiRNA, C) P1/FAMꢀsiRNA, D) P2/FAMꢀsiRNA, E) P3/FAMꢀ
siRNA and F) P4/FAMꢀsiRNA. P1: mPEGꢀbꢀP(CBꢀ{gꢀCation chain}ꢀcoꢀLA), P2: mPEGꢀbꢀP(CBꢀ{gꢀ
Cation chain; gꢀMorph}ꢀcoꢀLA), P3: mPEGꢀbꢀP(CBꢀ{gꢀCation chain; gꢀChol}ꢀcoꢀLA) and P4: mPEGꢀbꢀ
P(CBꢀ{gꢀCation chain; gꢀChol; gꢀMorph}ꢀcoꢀLA).
as a positive control, naked FAMꢀsiRNA as a negative control and four FAMꢀsiRNA
nanoplexes (P1/FAMꢀsiRNA, P2/FAMꢀsiRNA, P3/FAMꢀsiRNA and P4/FAMꢀsiRNA)
were taken as test samples. In 4T1 cells, the percentage transfection efficiency was found
to be 76.61%, 67.86%, 72.96%, 99.82% for copolymers P1, P2, P3 and P4, respectively
while lipoplexes (lipofectamineꢀ2000®/FAMꢀsiRNA) and naked FAMꢀsiRNA showed
transfection efficiency equivalent to 83.74% and 9.55% respectively (Figure 6). Further
in MCFꢀ7 cells the percentage transfection was found to be 36.69%, 39.10%, 42.34% and
50.82% for copolymers P1, P2, P3 and P4 respectively. Lipoplexes (lipofectamineꢀ
2000®/FAMꢀsiRNA) and naked FAMꢀsiRNA showed transfection efficiency of 53.94%
and 4.09% respectively (Figure 7).
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Figure 8. Intracellular localization of FAMꢀsiRNA (green) in 4T1 Cells after treatment with (A and C) FAMꢀ
siRNA/lipoplexes and (B and D) FAMꢀsiRNA/nanoplexes at 1 h and 2h, E1 and E2) colocalization analysis and
scattergram between green pixel intensity (FAMꢀsiRNA) and red pixel intensity (Lysosotracker: red) image of
cells treated with siRNA/lipoplexes, F1 and F2) colocalization analysis and scattergram between green pixel
intensity (FAMꢀsiRNA) and red pixel intensity (Lysosotracker: red) image of cells treated with FAMꢀ
siRNA/nanoplexess. Scale bar represents 10 ꢁm.
Endo/lysosomal escape
Intracellular trafficking and endosomal release of FAMꢀsiRNA from the
nanoplexes was studied using confocal microscopy (Figure 8). LysoTracker Red DNDꢀ
99 and DAPI dyes were used to stain the acidic vesicles and nucleus respectively. It was
observed that both nanoplexes and lipoplexes were able to deliver FAMꢀsiRNA (green
fluorescence) into the 4T1 cells within 1 h. In case of nanoplexes, discrete punctuate
spots were observed along the cell membrane. For lipoplexes diffused green fluorescence
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was observed in 1 h and 2 h samples. The colocalization was also analysed by
determining the overlap of the pixels of green fluorescence with red fluorescence. Percent
of volume colocalized for lipoplexes was 22.6% while in case of nanoplexes it was only
0.5% indicating no colocalization of FAMꢀsiRNA (green) with the acidic vesicles.
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Figure 9. Cytotoxicity assay of miRꢀ34a nanoplexes in A) 4T1 and B) MCFꢀ7 cells. Data were
presented as mean of three experiments in both cell lines. Statistical analysis were performed
using one way ANOVA followed by Tukey’s multiple comparison test. ***P < 0.05 was
considered as statistically significant.
Cytotoxicity assay miR-34a nanoplexes
miRꢀ34a is a wellꢀknown tumor suppressor and plays an important role in
cancer cells proliferation.21 Cellular cytotoxicity of miRꢀ34a nanoplexes was determined
in 4T1 and MCFꢀ7 breast cancer cells. 4T1 is a murine mammary carcinoma cell line
from a BALB/cfC3H mouse that represents tripleꢀnegative breast cancer and serves as
suitable model to test miRꢀ34a nanoplexes. Further, the sequence of hsaꢀmiRꢀ34aꢀ5p
mature sequence is similar to mmuꢀmiRꢀ34aꢀ5p mature sequence (www.mirbase.org).
Cells were treated with 20 picomole of miRꢀ34a nanoplexes for 48 h, after that the cell
viability was measured by MTT assay. Blank nanoplexes and mirVana™ miRNA Mimic
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#1 Negative control nanoplexes were kept as control and showed no cellular cytotoxicity,
while miRꢀ34a nanoplexes showed a significant decrease in the cancer cell viability in
both 4T1 (28%) and MCFꢀ7 (34%) cells as shown in Figure 9.
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Apoptosis assay
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Apoptosis in 4T1 and MCFꢀ7 cells was evaluated after 24 h treatment with miRꢀ
34a nanoplexes by flow cytometry. In FACS plot between FITCꢀA on the xꢀaxis and
propidium iodide on the yꢀaxis, the viable cells showed annexin V(ꢀ) and PI (ꢀ) (Q3 lower
(ꢀ)
left quadrant) whereas the cells in early phase apoptosis showed annexin V(+) and PI
(Q4 at the lower right side) and late phase apoptosis showed annexinV(+) and PI(+) (Q2 at
upper right) while necrotic cells only show PI(+) (Q1 at upper left side). It was observed
that in 4T1 cells, miRꢀ34a nanoplexes induced 40.66% apoptosis (23.12% early and
17.54% late stage) as compared to 2.54% apoptosis (0.99% early and 1.55% late stage)
Figure 10. Flow cytometric analysis of apoptosis in A) 4T1 and B) MCFꢀ7 cells after treatment
with blank nanoplexes, mirVana™ miRNA Mimic #1 Negative control (NC miRNA)
nanoplexes and miRꢀ34a nanoplexes. All nanoplexes were prepared using mPEGꢀbꢀP(CBꢀ{gꢀ
Cation chain; gꢀChol; gꢀMorph}ꢀcoꢀLA) copolymer.
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for NC miRNA nanoplexes and 3.57% apoptosis (1.27% early and 2.30% late stage) for
blank nanoplexes. Similar results were obtained in MCFꢀ7 cells wherein miRꢀ34a
nanoplexes induced 41.77% apoptosis (24.79% early and 16.98% late stage) as compared
to 2.89% apoptosis (1.93% early and 0.96% late stage) for NC miRNA nanoplexes and
3.88% apoptosis (2.29% early and 1.59% late stage) for blank nanoplexes (Figure 10).
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DISCUSSION
miRNAs are known to be involved in cancer progression, invasion and metastasis
wherein their levels are either upregulated or down regulated. miRNAs as therapeutic tool
in cancer has been explored by several research groups which have shown improved
outcome in terms of increased cytotoxicity, decreased invasion and metastasis.2, 8, 17, 40
Delivery strategies including nanoplexes,41 polyplexes,14 lipoplexes,42 micelleplexes15, 43
and dendriplexes44 have been reported for miRNA delivery since naked miRNAs could
not penetrate the bilayer lipidic membrane because of high molecular weight, hydrophilic
and anionic nature and instability in biological milieu. Apart from effectively complexing
miRNAs and protecting them from degradation, another important requirement for ideal
delivery system is that they should be able to improve the uptake of miRNA by cancer
cells followed by their successful endosomal escape and release into the cytoplasm so
that they can get into the RISC assembly for effective mRNA degradation and/or
translation repression.45
Among various miRNA delivery systems, cationic amphiphilic copolymers offer
advantages due to their selfꢀassembly, thermodynamic stability and lower particle size.
Further copolymers could be tailored to improve the delivery of miRNA therapeutics. In
a recent study, Kumar et al. prepared nanoplexes of miRꢀlet7b and a cationic copolymer,
mPEGꢀbꢀPCCꢀgꢀDCꢀgꢀTEPA at N/P at 16/1, that showed an average particle size of
80±10 nm.19 In another study,46 Zhu et al. delivered VEGF siRNA and paclitaxel for
prostate cancer by using biodegradable cationic nanoplexes of PDMAEMAꢀPCLꢀ
PDMAEMA triblock copolymers with micelle size of 53.6 to 132.2 nm.46
Successful in vivo delivery of a miRNAs mandates a multifunctional carrier
system which can ensure effective complexation with the miRNA (due to its cationic
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charge) along with ability to escape from endosomes and releasing it into the cytoplasm.
In the present study, we have designed amphiphilic cationic copolymers with different
ligands
viz.
cholesterol,
N,Nꢀdimethyldipropylenetriamine
and
4ꢀ(2ꢀ
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aminoethyl)morpholine attached on the hydrophobic block of the copolymer to enhance
the complexation ability, transfection efficiency and endosomal escape. All synthesized
polymers showed selfꢀassembly into nanoplexes in size ranging from 108 to 180 nm
depending upon the ligand attached to them. For selfꢀassembly, double emulsion method
yielded a smaller particle size and PDI as compared to film hydration method since high
hydrophobicity of the copolymer does not allow complete redispersion of the film
resulting in higher polydispersity.
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The synthesized copolymers were first screened for their complexation ability
with the miRꢀ34a followed by transfection efficiency in cancer cells. Complexation at
lower N/P ratios is preferred since higher polymer concentration could result in cellular
cytotoxicity. Use of different cationic ligands including tetraethylenepentamine, spermine
and N,Nꢀdimethyldipropylenetriamine have been reported earlier wherein it was shown
that copolymers containing N,Nꢀdimethyldipropylenetriamine effectively complexes
miRNA at a N/P ratio of 4:1.18 Our results indicate an efficient complexation with the
miRꢀ34a at a N/P ratio of 2:1 for copolymer P4 (Figure 5C). Further, presence of
endosomal escape moieties26 (such as imidazole, morpholine) could lead to efficient
escape of the miRNA from endosomes. Putnam et al. synthesized a polyꢀlꢀlysineꢀgraftedꢀ
imidazole acetic acid for effective gene transfer with lower cytotoxicity.47 In this report,
authors hypothesized that the balance between cationic amines with endosomal escaping
moieties leads to efficient gene transfer with lower cytotoxicity. A weakly basic
morpholino group could also have proton sponge effect, essential for endosomal
release.26 In our copolymer, N, Nꢀdimethyldipropylenetriamine cationic chain was
incorporated for complexing with the miRꢀ34a by charge interactions resulting in
nanoplex formation while morpholine helps in endosomal escape of the nanoplexes.
Cholesterol is one of the components of cell membrane so its incorporation in the
structure of gene carrier could help in improving biocompatibility and cellular uptake of
the carrier. Cheng et al. reported a cholesterol grafted bioreducible (polyamidoamine)
polymer for delivery of siRNA which resulted in a stable siRNA nanoplexes with higher
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transfection ability and endosomal escape.21 Frere et al. have also synthesized a
biocompatible polycarbonate polymer containing morpholine and guanidinium for
increasing the transfection efficiency, endosomal escape and complexation ability with
siRNA.14 In the present study, we prepared nanoplexes by using cationic copolymers with
FAM labeled siRNA and compared with lipofectamine-2000® (cationic Lipid) as a
positive control for in vitro transfection in cancer cells. The nanoplexes prepared using
copolymer containing both morpholine and cholesterol have showed efficient transfection
in 4T1 and MCFꢀ7 cells as compared to lipofectamineꢀ2000® (Figure 6 and 7). Higher
transfection by this copolymer could be correlated with the zeta potential of the
nanoplexes as reported earlier by Huang et al. wherein better cellular uptake was
observed with formulations having higher zeta potential.24
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In order to study the endosomal escape of the nanoplexes, LysoTracker Red DND
99 dye was used to label the acidic vesicles in the cell and colocalization was studied
between green fluorescence from FAMꢀsiRNA and red fluorescence from
endo/lysolomes. Similar studies were also reported by Nelson et al. for colocalization of
pH responsive siRNA polyplexes with acidic vesicles wherein polyplexes showed
significantly lower colocalization relative to lipoplexes prepared using lipofectamine-
2000®.48 Our data also suggest an efficient endosomal escape of nanoplexes and thus
overcome the hindrance of endoꢀlysosomal barrier in delivery. Another important aspect
of miRNA delivery is the safety of the carrier system. In general cationic polymers such
as polyethylenimine (PEI) were shown to be cytotoxic thus limiting their translational
potential.49 The developed copolymers showed negligible cellular cytotoxicity towards
4T1 and MCFꢀ7 cells and were nonꢀhemolytic. In order to access the in vitro efficacy of
the miRꢀ34a nanoplexes, cytotoxicity and apoptosis assays were performed. miRꢀ34a has
been used as a master tumour suppressor miRNA owing to its role in several cancers
including breast cancer. Zhao et al. have earlier reported that its overexpression
suppresses the MCFꢀ7 cell proliferation and cell viability.7 We also observed a significant
decrease in the cell viability of 4T1 and MCFꢀ7 cells after treatment with miRꢀ34a
nanoplexes. While negative control miRNA nanoplexes and blank polyplexes, showed no
effect on cell viability indicating their nonꢀtoxic and biocompatible nature. Further,
apoptosis assay showed miRꢀ34a nanoplexes effectively suppressed the tumour cell
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growth by inducing early and late phase apoptosis in breast cancer cells. In summary, the
cationic copolymers containing morpholine and cholesterol in the backbone could be
used as potential nonꢀviral carriers for delivering miRꢀ34a. They selfꢀassemble to form a
stable nanoplexes of <200 nm at a low N/P ratio and exhibit improved transfection,
endosomal escape and ability to inhibit cancer cell growth.
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Acknowledgements
The funding support from Department of Science and Technology (DST),
Government of India to DC through young scientist award (YSS/2014/000521) is duly
acknowledged. The authors gratefully acknowledge Advanced Instrumentation Research
Facility (AIRF), Jawaharlal Nehru University (JNU), New Delhi, India for providing
instrumental support for Confocal Laser Scanning Microscopy (CLSM). We would also
like to thank Mr. Ashok Kumar Sahu from AIRF, JNU, New Delhi for his help in taking
Confocal images. Also we would like to thank Sophisticated Test & Instrumentation
Centre (STIC), Cochin University of Science and Technology, Kerala, India for
elemental analysis.
Conflicts of interest
There are no conflicts to declare.
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